Photon reducing agents for fluorescence assays

ABSTRACT

The present invention provides a method for reducing undesirable light emission from a sample using at least one photon producing agent and at least one photon reducing agent (e.g. dye-based photon reducing agents). The present invention further provides a method for reducing undesirable light emission from a sample (e.g., a biochemical or cellular sample) with at least one photon producing agent and at least one collisional quencher. The present invention also provides a method for reducing undesirable light emission from a sample (e.g., a biochemical or cellular sample) with at least one photon producing agent and at least one quencher, such as an electronic quencher. The present invention also provides a system and method of screening test chemicals in fluorescent assays using photon reducing agents. The present invention also provides compositions, pharmaceutical compositions, and kits for practicing these methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/398,744, filed Mar. 5, 2009, now U.S. Pat. No. 7,785,536, which is acontinuation of U.S. patent application Ser. No. 11/047,074, filed Jan.31, 2005, abandoned, which is a continuation of U.S. patent applicationSer. No. 09/759,629, filed Jan. 12, 2001, now U.S. Pat. No. 7,067,324,which is a continuation of U.S. patent application Ser. No. 09/122,477,filed Jul. 23, 1998, now U.S. Pat. No. 6,221,612, which claims thebenefit of priority under 35 U.S.C. §119(e) to U.S. provisionalapplication No. 60/054,519, filed Aug. 1, 1997.

TECHNICAL FIELD

The present invention generally relates to methods and compositions forreducing undesired light from an assay sample, particularly fluorescenceassays in living cells.

BACKGROUND

Cell-based assays are commonly used for drug discovery to screen largenumbers of test chemicals for potential therapeutic activity. Typically,the cells contain a target, such as a protein. Test-chemicals, such ascandidate ligands for a target protein, are screened for modulatingactivity of a target. Screening relies on a detectable change in aproperty of a cell that faithfully reports target activity in thepresence of a test chemical. Many assays use optical methods to detectsuch activities. Fluorescence detection methods are particularlypowerful tools in this regard, because fluorescence detection methodscan be sensitive. Many different types of fluorescent probes areavailable for such assays, including fluorescent probes that act asenzyme substrates, labels for proteins and nucleic acids, indicators ofintra-cellular ions, and sensors of membrane voltage.

Despite the recent plethora in available fluorescent tools for assays,fluorescence based assays can be plagued by undesirable, and sometimesintolerable, levels of background fluorescence. For example, solutionfluorescence may increase the background fluorescence of the assaysample. Solution fluorescence can obscure a desired signal associatedwith a fluorescent probe. Solution fluorescence can arise from manysources, including fluorescent probe degradation, targets, cells,various solution components, and test chemicals.

In cell-based assays recently developed by one of the inventors of thepresent invention, solution fluorescence can give rise to lower signalto noise ratios. These cell-based assays utilize a membrane permeablesubstrate specific for beta-lactamase, a bacterial enzyme that is notnormally expressed in mammalian cells. The substrate diffuses into thecell and is trapped inside the cell by the action of intracellularesterases.

If a cell expresses a beta-lactamase reporter gene, the expressed enzymewill cleave the substrate. Before cleavage the substrate fluorescesgreen and after cleavage the substrate fluoresces blue. When such assaysare used for high-throughput screening, increasing the signal to noiseratio can be advantageous because it increases the sensitivity of thescreening system and reliability of the data. Solution fluorescence,however, often thwarts achieving advantageous signal to noise ratios.Solution fluorescence from test chemicals, substrate in the solution,and other solution components that bath the cells contribute tobackground fluorescence.

The present inventors recognized that membrane compartment assays, suchas cell-based assays, that use optical methods could be improved byreducing unwanted light emitted from the solution bathing the membranecompartments, particularly solution fluorescence. The present inventorsinvestigated different washing and incubation methods in an attempt toincrease dye loading and retention while reducing solution fluorescence.Although the inventors could reduce solution fluorescence, suchmanipulations were cumbersome and time consuming.

Consequently, the present inventors developed compositions and methodsfor reducing the emission undesired light from solutions in membranecompartment assays that did not solely rely on washing or incubationmethods. Such compositions and methods are much more applicable tohigh-throughput screening than improvements to washing and incubationmethods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a diagram of fluorescence emission from a solution without aphoton reducing agent.

FIG. 1B is a diagram of fluorescence emission from a solution with aphoton reducing agent.

FIG. 2 shows the ability of phenol red to reduce the emission offluorescence from a solution containing coumarin.

FIG. 3 shows the ability of phenol red to reduce the emission offluorescence from a solution containing coumarin the presence andabsence of methanol.

FIG. 4 shows the dependence of emission of fluorescence from a solutionon candidate photon reducing agent concentration.

FIG. 5 shows the dependence of emission of fluorescence from a solutioncontaining coumarin in the presence of various photon reducing agents.

FIG. 6 shows the dependence of emission of fluorescence from a solutioncontaining fluorescein in the presence of various colored photonreducing agents.

FIG. 7 shows the dependence of emission of fluorescence from a solutioncontaining rhodamine in the presence of various colored photon reducingagents.

FIG. 8 shows residual CCF2 solution fluorescence as a function ofcolored photon reducing agent concentration.

FIG. 9A, FIG. 9B, and FIG. 9C show the reduction of solutionfluorescence using non-dye based photon agents that electronicallyinteract with a photon producing agent.

FIG. 10A, FIG. 10B, and FIG. 10C show coumarin, fluorescence isattenuated by phenol red at various pathlengths.

FIG. 11 shows that photon reducing agents reduce the fluorescenceemission in unwashed cells and yields signals comparable to signals fromwashed cells.

FIG. 12 summarizes the results of photon reducing agent toxicitytesting.

FIG. 13 shows the ability of cells treated with photon reducing agentsto express beta-lactamase after stimulation with an appropriate agonist.

FIG. 14A and FIG. 14B show that photon reducing agent sets can reduceundesired fluorescence better than single dye-based photon reducingagents.

SUMMARY

The present invention provides for a method of reducing light emission,such as undesirable light, from a sample, such as a solution. The methodcan be used with fluorescent assays that are often hampered by solutionfluorescence that interferes with detecting a desired signal from thesample. Membrane compartment based assays, such as cell-based assays,typically exhibit undesirable background fluorescence from probes, testchemicals, or other solution components that can interfere with desiredsignal detection. To overcome these problems, the inventors devised amethod to reduce undesired light emitted from the sample by adding aphoton reducing agent to the sample. These methods can be used, forexample, to identify a chemical with a biological activity. The presentinvention also includes a therapeutic composition identified by suchmethods and a system to perform such methods and to identify a chemicalwith a toxicological or bioavailability activity.

The present invention also provides a composition of matter comprising amembrane compartment that is in physical or optical contact with a solidsurface, such as a surface that can transmit light, and an aqueoussolution with at least one photon reducing agent. The solid surface canbe, for example, a well of a multi-well platform, such as a microtiterplate. Optionally, the membrane compartment need not be in contact witha solid surface. In this aspect of the present invention, the membranecompartment can be within a drop or droplet such as they are generatedduring FACS procedures.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein, and the laboratory procedures in spectroscopy, drugdiscovery, cell culture, and molecular genetics, described below, arethose well known and commonly employed in the art. Standard techniquesare typically used for signal detection, recombinant nucleic acidmethods, polynucleotide synthesis, and microbial culture andtransformation (e.g., electroporation, and lipofection). The techniquesand procedures are generally performed according to conventional methodsin the art and various general references (see generally, Sambrook etal. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; and Lakowicz, J. R.Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983))for fluorescence techniques, each of which are incorporated herein byreference) which are provided throughout this document. Standardtechniques are used for chemical syntheses, chemical analyses, andbiological assays. As employed throughout the disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

“Fluorescent donor moiety” refers to the radical of a fluorescentcompound that can absorb energy and is capable of transferring theenergy to an acceptor, such as another fluorescent compound or anotherpart of the fluorescent compound. Suitable donor fluorescent compoundsinclude, but are not limited to, coumarins and related dyes, xanthenedyes such as fluoresceins, rhodols, and rhodamines, resorufins, cyaninedyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalichydrazides such as luminol, and isoluminol derivatives,aminophthalimides, aminonaphthalimides, aminobenzofurans,aminoquinolines, dicyanohydroquinones, and fluorescent europium andterbium complexes and related compounds.

“Quencher” refers to a molecule or part of a compound that is capable ofreducing the emission from a fluorescent moiety. Such reduction includesreducing the light after the time when a photon is normally emitted froma fluorescent moiety. Quenching may occur by any of several mechanisms,including fluorescence resonance energy transfer, photoinduced electrontransfer, paramagnetic enhancement of intersystem crossing, Dexterexchange coupling, and excitation coupling, such as the formation ofdark complexes.

“Acceptor” refers to a quencher that operates via energy transfer.Acceptors may re-emit the transferred energy as fluorescence. Examplesof acceptors include coumarins and related fluorophores, xanthenes suchas fluoresceins, rhodols, and rhodamines, resorufins, cyanines,difluoroboradiazaindacenes, and phthalocyanines. Other chemical classesof acceptors generally do not re-emit the transferred energy. Examplesinclude indigos, benzoquinones, anthraquinones, azo compounds, nitrocompounds, indoanilines, and di- and triphenylmethanes.

“Binding pair” refers to two moieties (e.g. chemical or biochemical)that have an affinity for one another. Examples of binding pairs includeantigen/antibodies, lectin/avidin, target polynucleotide/probeoligonucleotide, antibody/anti-antibody, receptor/ligand, enzyme/ligandand the like. “One member of a binding pair” refers to one moiety of thepair, such as an antigen or ligand.

“Dye” refers to a molecule or part of a compound that absorbsfrequencies of light, including, but not limited to, ultraviolet light.The terms “dye” and “chromophore” are synonymous.

“Fluorophore” refers to a chromophore that fluoresces.

“Membrane-permeant derivative” refers to a chemical derivative of acompound that has enhanced membrane permeability compared to anunderivativized compound. Examples include ester, ether and carbamatederivatives. These derivatives are made better able to cross cellmembranes (i.e. membrane permeant) because hydrophilic groups are maskedto provide more hydrophobic derivatives. Also, masking groups aredesigned to be cleaved from a precursor (e.g., fluorogenic-substrateprecursor) within a cell to generate the derived substrateintracellularly. Because the substrate is more hydrophilic than themembrane permeant derivative, it becomes trapped within the cell.Membrane-permeant and membrane-impermeant are relative terms based onthe permeability characteristics of a compound and a chemical derivativethereof.

“Isolated polynucleotide” refers a polynucleotide of genomic, cDNA, orsynthetic origin, or some combination there of, which by virtue of itsorigin the “isolated polynucleotide” (1) is not associated with the cellin which the “isolated polynucleotide” is found in nature, or (2) isoperably linked to a polynucleotide which it is not linked to in nature.

“Isolated protein” refers a protein, encoded by cDNA, recombinant RNA,or synthetic nucleic acids, or some combination thereof, which by virtueof its origin the “isolated protein” (1) is not associated with proteinsthat it is normally found with in nature, (2) is isolated from the cellin which it normally occurs, (3) is isolated free of other proteins fromthe same cellular source (e.g. free of human proteins), (4) is expressedby a cell from a different species, or (5) does not occur in nature.“Isolated naturally occurring protein” refers to a protein which byvirtue of its origin the “isolated naturally occurring protein” (1) isnot associated with proteins that it is normally found with in nature,or (2) is isolated from the cell in which it normally occurs, or (3) isisolated free of other proteins from the same cellular source, e.g. freeof human proteins.

“Polypeptide” as used herein as a generic term to refer to nativeprotein, fragments, or analogs of a polypeptide sequence. Hence, nativeprotein, fragments, and analogs are species of the polypeptide genus.

“Naturally-occurring” as used herein, as applied to an object, refers tothe fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. For example, a control sequence “operably linked” to acoding sequence is linked (for example, ligated) in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequences, such as when the appropriatemolecules (e.g., inducers and polymerases) are bound to the control orregulatory sequence(s).

“Control sequence” refers to polynucleotide sequences that are necessaryto effect the expression of coding and non-coding sequences to whichthey are linked (for example, ligated). The nature of such controlsequences differs depending upon the host organism. In eukaryotes, suchcontrol sequences generally include enhancers, promoters, ribosomalbinding sites, and transcription termination sequences. In prokaryotes,generally, such control sequences include promoters and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, components whose presence can influence theexpression of a gene, and can include additional components whosepresence is advantageous, for example, leader sequences and fusionpartner sequences (for example, sequences encoding a fusion protein).

“Polynucleotide” refers to a polymeric form of nucleotides of at least10 bases in length, either ribonucleotides or deoxynucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA.

“Corresponds to” refers to a sequence that is homologous (i.e., isidentical, not strictly evolutionarily related) to all or a portion of areference sequence.

“Membrane compartment” refers to a semi-permeable material (for example,a biological membrane, vesicle, cell (for example, prokaryotic oreukaryotic, such as mammalian, such as human), liposome, envelope of avirus, or the like, surrounding a volume of aqueous fluid, such asintracellular fluid.

“Modulation” refers to the capacity to either enhance or inhibit afunctional property of biological activity or process (e.g., enzymeactivity or receptor binding). Such enhancement or inhibition may becontingent upon the occurrence of a specific event, such as activationof a signal transduction pathway, and can be exhibited only inparticular cell types.

The term “modulator” refers to a chemical compound (naturally occurringor non-naturally occurring), such as a biological macromolecule (e.g.,nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal (particularly mammalian, including human) cells or tissues.Modulators are evaluated for potential activity as inhibitors oractivators (directly or indirectly) of a biological process or processes(e.g., agonist, partial antagonist, partial agonist, inverse agonist,antagonist, antineoplastic agents, cytotoxic agents, inhibitors ofneoplastic transformation or cell proliferation, cellproliferation-promoting agents, and the like) by inclusion in screeningassays described herein. The activity of a modulator may be known,unknown or partially known.

The term “test chemical” refers to a chemical to be tested by one ormore screening method(s) of the invention as a putative modulator. Atest chemical can be any chemical, such as an inorganic chemical, anorganic chemical, a protein, a peptide, a carbohydrate, a lipid, or acombination thereof.

The terms “label” or “labeled” refers to incorporation of a detectablemarker. For example, by incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). Various methods of labeling polypeptides and glycoproteins areknown in the art and may be used. Examples of labels for polypeptidesinclude, but are not limited to, the following: radioisotopes (e.g., ³H,¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels or a product of a reporter gene(e.g., horseradish peroxidase, beta-galactosidase, beta-latamase,luciferase, and alkaline phosphatase), other labels such aschemiluminescent labels, biotinyl groups, or predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, and epitope tags). In some embodiments, labels are attached byspacer arms of various lengths to reduce potential steric hindrance.

“Fluorescent label” refers to a fluorescent moiety incorporated onto orwithin a chemical structure having desirable properties, such as bindingwith a target or attaching to a polypeptide of biotinyl moieties thatcan be detected by avidin (e.g., streptavidin containing a fluorescentlabel or enzymatic activity that can be detected by fluorescencedetection methods). Various methods of fluorescently labelingpolypeptides, glycoproteins and other moieties are known in the art andmay be used. Examples of labels for polypeptides include, but are notlimited to dyes (e.g., FITC and rhodamine), intrinsically fluorescentproteins, and lanthanide phosphors. In some embodiments, labels areattached by spacer arms of various lengths to reduce potential sterichindrance.

“Photon reducing agent” refers to a molecule or particle, such as acolloidal particle, that reduces that amount of light emitted fromanother molecule in a sample or reduces the amount of light that excitesanother molecule in a sample. Typically, a photon reducing agent reducesthe amount of light emitted from another molecule in a sample by havingan absorption spectrum that overlaps with the absorption, excitation, oremission spectrum of a molecule that produces photons. Alternatively,some photon reducing agents may engage in energy transfer (e.g.,fluorescence resonance energy transfer (FRET)) with a photon producingagent that prevents or alters the emission of light from the photonproducing agent.

“Photon producing agent” refers to a molecule that can emit photons.Typically, a photon producing agent produces photons by absorbing lightat one wavelength and emitting light of another wavelength.

“Reporter gene” refers to a nucleotide sequence encoding a protein thatis readily detectable either by its presence or activity, including, butnot limited to, luciferase, green fluorescent protein, chloramphenicolacetyl transferase, beta-galactosidase, secreted placental alkalinephosphatase, beta-lactamase, human growth hormone, and other secretedenzyme reporters. Generally, reporter genes encode a polypeptide nototherwise produced by a host cell, which is detectable by analysis ofthe cell or a population of cells, e.g., by the direct fluorometric,radioisotopic, optical or spectrophotometric analysis of the cell or apopulation of cells and preferably without the need to kill the cellsfor signal analysis. Preferably, the reporter gene encodes an enzymethat produces a change in at least one fluorescent property of or in thehost cell. The at least one fluorescent property is preferablydetectable by qualitative, quantitative or semi-quantitative functionmethods, such as the detection of transcriptional activation. Exemplaryenzymes include esterases, phosphatases, proteases (for example, tissueplasminogen activator or urokinase) and other enzymes (such asbeta-lactamase or luciferase or sugar hydrolases, such asbeta-galactosidase) whose function can be detected by appropriatechromogenic or fluorogenic substrates known to those skilled in the art.

“Plate” refers to a multi-well plate, unless otherwise modified in thecontext of its usage.

“Sample” refers to any fluid, solid, jelly, emulsion, slurry, or amixture thereof that contains a membrane compartment. A sample ispreferably an aqueous solution that contains a cell, such as aeukaryotic cells, such as a mammalian cell, such as a human cell.

“Signal transduction detection system” refers to a system for detectingsignal transduction across a cell membrane, typically a cell plasmamembrane. Such systems typically detect at least one activity orphysical property directly or indirectly associated with signaltransduction. For example, an activity or physical property directlyassociated with signal transduction is the activity or physical propertyof either the receptor (e.g., GPCR), or a coupling protein (e.g., a Gαprotein). Signal transduction detection systems for monitoring anactivity or physical property directly associated with signaltransduction, include the detection of GTPase activity andconformational changes of members of the signal transduction system. Anactivity or physical property indirectly associated with signaltransduction is the activity or physical property produced by a moleculeother than by either the receptor (e.g., GPCR), or a coupling protein(e.g., a Gα protein) associated with receptor (e.g., GPCR), or acoupling protein (e.g., a Gα protein). Such indirect activities andproperties include changes in intracellular levels of molecules (e.g.,ions (e.g., Ca⁺⁺, Na⁺ or K⁺), second messenger levels (e.g., cAMP, cGMPand inostol phosphate)), kinase activities, transcriptional activity,enzymatic activity, phospholipase activities, ion channel activities andphosphatase activities. Signal transduction detection systems formonitoring an activity or physical property indirectly associated withsignal transduction include, for example, transcriptional-based assays,enzymatic assays, intracellular ion assays and second messenger assays.

“Solution fluorescence” refers to fluorescence from a fluorophore in asolution. For instance the fluorophore may be a test chemical (or acomponent associated with the test compound or a component of themeasurement system itself) in an assay buffer. Solution fluorescence isone component of background fluorescence. Background fluorescence mayarise from other sources, such as assay vessels (e.g., microtiterplates), optical relay systems and backscatter.

A “target” refers to any biological entity, such as a protein, sugar,carbohydrate, nucleic acid, lipid, a cell or population of cells or anextract thereof, a vesicle, or any combination thereof.

“Transmittance” refers to the fraction of incident light that passesthrough a medium at a given wavelength. It can also be considered theratio of radiant power transmitted through a medium to the radiant powerincident on the medium at a particular wavelength.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(ed. Parker, S., 1985), McGraw-Hill, San Francisco, incorporated hereinby reference).

Introduction

The present invention recognizes for the first time that the addition ofa photon reducing agent can decrease undesired light emission from asample. Typically the sample comprises a membrane compartment, using aphoton producing agent. The present invention also recognizes for thefirst time that solution fluorescence in cell-based assays can bereduced by adding a photon reducing agent, such as a dye, to thesolution bathing the cells. Aspects of the invention are based, in part,on the counter-intuitive finding that the addition of a chemical having“color” can improve fluorescence assay measurements by reducing solutionfluorescence while retaining signal fluorescence from a separate aqueouscompartment. The advantages of the present invention include: 1)increasing the signal to noise ratio in assays utilizing membranecompartments, 2) decreasing assay variability, 3) reducing assay time,4) reducing assay manipulation (especially compared to assays withwashing steps), and 5) minimizing solution fluorescence.

As a non-limiting introduction to the breadth of the invention, theinvention includes several general and useful aspects, including:

-   -   (1) a method for reducing undesirable light emission from a        sample (e.g., a biochemical or cellular sample) with at least        one photon producing agent by using at least one photon reducing        agent (e.g. dye-based photon reducing agents),    -   (2) a method for reducing undesirable light emission from a        sample (e.g., a biochemical or cellular sample) with at least        one photon producing agent by using at least one collisional        quencher,    -   (3) a method for reducing undesirable light emission from a        sample (e.g., a biochemical or cellular sample) with at least        one photon producing agent by using at least one quencher, such        as an electronic quencher,    -   (4) a method of screening test chemicals in fluorescent assays        using photon reducing agents,    -   (5) compositions, therapeutic compositions and kits for        practicing (1), (2), (3), (4), and (5),    -   (6) a system for identifying the compositions of (6), and    -   (7) a method of identifying a chemical with toxicological        activity.

These aspects of the invention and others described herein, can beachieved by using the methods and compositions of matter describedherein. To gain a full appreciation of the scope of the invention, itwill be further recognized that various aspects of the invention can becombined to make desirable embodiments of the invention. For example,the invention includes a method for reducing background fluorescenceusing dye-based photon reducing agents in assays to identify testchemicals that modulate target proteins. Such combinations result inparticularly useful and robust embodiments of the invention.

Methods for Reducing Undesired Light Emission from a Sample Using atLeast One Photon Reducing Agent

The invention provides for a method of reducing undesirable lightemission from a sample. The method can be used with fluorescence assaysthat are often hampered by solution fluorescence that interferes withdetecting a desired signal from the sample. Cell-based assays typicallyemit solution fluorescence from probes or test chemicals that canobscure desired signal detection. To avoid these problems, the inventorsdevised a method to reduce undesired light emitted from the sample byadding a photon reducing agent to the sample. As described more fullyherein, “photon reducing agent” refers to a molecule that reduces thatamount of light from a sample by another molecule.

The method comprises the steps of contacting a sample with at least onephoton reducing agent and detecting an optical signal from a photonproducing agent. The sample typically comprises a membrane-enclosedcompartment in contact with a solid surface that can pass (e.g.transmit) light. The membrane compartment usually includes at least onephoton producing agent. As described more fully herein, “photonproducing agent” refers to a molecule that emits light. The photonproducing agent is typically located either within an aqueous interiorof the membrane-enclosed compartment or in association with the membraneor some other component of the membrane compartment (e.g., a cellularorganelle). The photon reducing agent is typically located in an aqueoussolution that contacts the outer surface of the membrane compartment.The aqueous solution that contacts the outer surface also typicallycontains the source or sources of light (e.g., photon producing agents)that lead to unwanted light emission from a sample. Photon reducingagents reduce the light emitted from a sample that originates fromphoton producing agents in the aqueous solution.

Samples in need of a reduction in undesired light emission, as describedmore fully herein, are typically associated with fluorescent assays andrange from chemical or biochemical samples (e.g.,vesicles-compartmentalizing photon producing agents) to living cellsamples (e.g., cell-based assays using reporter genes). Such samplesoften have solution fluorescence that contributes to increasedbackground fluorescence that can either prevent the measurement of adesired signal or reduce the signal to noise ratio compared to detectionof the signal in the presence of a photon reducing agent.

Photon producing agents can be fluorescent protein, such as anAequorea-related fluorescent protein or a mutant thereof (see, forexample, U.S. Pat. No. 5,625,048 to Tsien, issued Apr. 29, 1997; WO96/23810 to Tsien et al., published Aug. 8, 1996; WO 97/28261 to Tsienet al., published Aug. 7, 1997; PCT/US97/12410 to Tsien et al, filedJul. 16, 1997; and PCT/US97/14593, filed Aug. 15, 1997), a fluorescentor fluorogenic enzymatic substrate (see, for example, WO 96/30540 toTsien et al., published Oct. 3, 1996), a member of a FRET pair, or candetect a voltage across a membrane of a cell (see, U.S. Pat. No.5,661,035 to Tsien et al., issued Aug. 26, 1997), an intracellular ionindicator, such as for calcium ions, such as Fluo3, Fura2, Indol, orfluorescent labels used in specific binding reactions, such asimmunoassays or receptor-ligand assays.

To reduce solution fluorescence, the invention utilizes aphoton-reducing agent. Photon reducing agents may be selected to reducelight from another molecule by a mechanism or mechanisms that allow forthe reduction of the emission of unwanted light emission from a sample.One class of photon reducing agents may absorb, and therefore reduce,the amount of unwanted light emitted from a sample comprising a photonproducing agent.

Desirable photon reducing agents typically have an absorption spectrumthat overlaps with the absorption, excitation or emission spectrum (or acombination thereof) of a photon producing agent. Alternatively, anotherclass of photon reducing agents may quench (e.g., by fluorescenceresonance energy transfer (FRET)) a photon producing agent. Otherquenching mechanisms or agents may be used, including collisionalquenchers, electronic quenching, particular quenching, exeplexformation, photo-induced electron transfer, paramagnetic or heavy-atomquenching leading to enhanced intersystem crossing. (see generally,Principles of Fluorescence Spectroscopy by Joseph R. Lakowicz. PlenumPress 1983). Other photon reducing agents are optical interferants thatcan reduce the amount of light emitted from a photon producing agent bylight scatter, refraction or reflectance. For example, particulatesreduce light emission from a photon producing agent, in part, by lightscattering. It is understood that reduced light emission from a photonproducing agent can result from many types of photon reducing agentsworking with different mechanisms. It is also understood that in certainapplications it will be desirable to select photon reducing agents thatreduce or decrease undesired light emission from a sample by more thanone mechanism. For instance, a photon reducing agent can be selectedthat reduces solution fluorescence by FRET and has an absorptionspectrum that overlaps with the absorption, excitation or emissionspectrum of a molecule that produces light. Selection of a photonreducing agent(s) is described more fully herein.

Typically, in a fluorescent assay, at least one photon reducing agentcan be selected that has an absorption spectrum that overlaps with theabsorption, emission or excitation spectrum of a photon producing agentlocated outside of a membrane compartment. In some instances the photonproducing agent may be located inside and outside the membranecompartment, such as with a membrane permeable sensor that leaks outthrough the membrane compartment and into the surrounding solution. Aphoton producing agent can also be free or bound inside a cell, such asa living cell that does not have a cell wall, such as a mammalian cell(such as a human cell. An assay may also include a second photonproducing agent in an aqueous solution surrounding the membranecompartment or at a site other than the site of desirable signalemission. The number of photon reducing agents in an assay typicallyranges from between 1 and 5, between 1 and 4, between 1 and 3, between 1and 2, and may include at least two or more or at least three or more.For example, the first photon producing agent may be a reporter genesubstrate or product located inside of a cell, and the second photonproducing agent may be a test chemical in the bathing solution.

Photon reducing agents can be readily selected for an absorptionspectrum that overlaps with the absorption, emission or excitationspectrum of a photon producing agent. As described herein, theabsorption spectra of a photon reducing agent can be readily measuredand compared to measured absorption, emission or excitation spectrum ofa known or expected photon producing agent. Such known or expectedphoton producing agents include, fluorescent reporter substrates,fluorescent labels, fluorescent membrane sensors, fluorescent proteins,test chemicals and intracellular analyte indictors (e.g., ionchelators). Methods known or developed in the art for measuring andcomparing absorption spectra can also be used to identify photonreducing agents. Light reducing dyes refer to photon reducing agentsthat have an absorption spectrum that overlaps with the absorption,emission or excitation spectrum of a photon producing agent.

When selecting a photon reducing agent, such as a light reducing dye, itis advantageous to compare the extent of its absorption spectrum overlapwith 1) the absorption, emission or excitation spectrum of a photonproducing agent in aqueous solution and 2) the absorption, emission orexcitation spectrum of the expected signal molecule in an assay sample.This comparison can aid in the selection of a photon reducing agent,such as a light reducing dye, by optimizing the spectral overlaps. Inaddition, it is desirable to select photon reducing agents with highextinction coefficients in order to reduce the amount of photon reducingagent needed for the desired effect.

Preferable photon reducing agents typically at least partially blockeither or both of the excitation or emission wavelengths of photonproducing agents. In doing so, preferable photon reducing agents reduceundesired light emission from a sample. Such preferable photon reducingagents can be determined by comparing the extinction coefficients ofcandidate photon reducing agents with the expected photon producingagents at the desired wavelength or range of wavelengths, by empiricalobservations, or by routine experimentation to select such desiredphoton producing agents using the methods of the present invention.Photon reducing agents can reduce the emission of undesired light from asample by at least about 10 percent, preferably at least about 30percent, more preferably at least about 50, and most preferably betweenabout 70 and 99 percent as compared to light emission from a sample or aparticular photon producing agent in the absence of a photon reducingagent.

Such photon producing agents and photon reducing agents can bedetermined, for example, by exciting a sample comprising a photonproducing agent and a photon reducing agent with light of a firstwavelength bandwidth and collecting the emission from the sample at asecond wavelength bandwidth. Preferably, the first wavelength bandwidthand the second wavelength bandwidth do not overlap, but they may.Preferable first wavelength bandwidths and preferable second wavelengthbandwidths can be determined by routine experimentation using methods ofthe present invention to determine such wavelength bandwidth ranges andoverlaps. Such bandwidths preferably include the appropriate excitationor emission peaks of at least one of the photon producing agent orphoton reducing agent, but that need not be the case because significantexcitation or emission can be obtained over a large portion of theappropriate excitation spectra or emission spectra.

Photon reducing agents are preferably provided at a workingconcentration in a sample between about 0.1 mM and about 10 mM and morepreferably between about 0.5 mM and 5 mM. When two or more photonreducing agents are present in a sample, the combined concentration ofthe photon reducing agents is preferably between about 0.1 mM and 10 mMand more preferably between about 0.5 mM and 5 mM. Photon reducingagents can increase the signal-to-noise ration of an assay by betweenabout 50% to about 100,000% or greater, and preferably between about500% and about 3,000%. The percent increase in signal-to-noise ratio(S/N) in the presence of a photon reducing agent (PRA) can be calculatedby the formula ((S/N in the presence of a PRA)/(S/N in the absence of aPRA))×100=percent increase in S/N.

Photon reducing agents also can be substantially impermeant to themembrane of a membrane compartment. Substantially impermeant, in thisinstance, means that under assay conditions, the concentration of thephoton reducing agent within the membrane compartment is less than 50%,preferably less than 30%, and most preferably less than 10% of theconcentration outside the membrane compartment.

Preferable photon reducing agents have a partition coefficient(octanol/water) equal to or less than CCF2/AM, at a pH between about 6and 8, preferably about pH 7, so that the photon reducing agentpreferably partitions in an aqueous solution rather than in ahydrophobic phase, such as a membrane (for CCF2/AM, see U.S. Pat. No.5,741,657 to Tsien et al., issued Apr. 21, 1998). Also, preferablephoton reducing agents have solubility in water of at least about 1 mMand preferably at least about 10 mM under assay conditions, such asbetween about 4° C. and 42° C., preferably between about 24° C. and 37°C. In addition, photon reducing agents are preferably should be moreimpermeant across a membrane compartment, such as a mammalian cell, thana photon producing agent used in an assay. Photon reducing agents can bea pH indicator dye and be dyes, such as azo dyes.

Preferable photon reducing agents also have an extinction coefficient ofbetween about 2,000 M⁻¹ cm⁻¹ to about 500,000 M⁻¹ cm⁻¹, preferablybetween about 10,000 M⁻¹ cm⁻¹ and 200,000 and more preferably greaterthan 10,000 M⁻¹ cm⁻¹ at a wavelength or range of wavelengths used in anassay.

In many instances the photon producing agent that leads to unwantedlight emitted from the sample will be the signal molecule in anon-desired location or compartment. Such instances typically occur whenthe signal molecule is present in the surrounding solution and reducingsolution fluorescence becomes desirable. For example, a fluorescentreporter that leaks out of a membrane compartment, such as a cell, willoften decrease the signal to noise ratio of the assay. When the photonproducing agent is the signal molecule, it is desirable to select alight reducing dye having an absorption spectrum that significantlyoverlaps with the absorption, emission or excitation spectrum of theexpected signal molecule. Preferably, such photon reducing agents can beidentified by determining the percentage overlap of spectrum asdetermined from the concentration and extinction coefficient of a photonproducing agent and a photon reducing agent at a desired wavelength orrange of wavelengths (see, Lakowicz, J. R. Principles of FluorescenceSpectroscopy, New York: Plenum Press (1983)). Generally, the wavelengthrange for such spectra is about 260 nm to about 900 nm, although narrowranges can be used, such as about 290 to 800 nm, and about 300 to 700nm. In addition it is advantageous to select a photon reducing agentwith an optical density or extinction coefficient sufficiently high tobe effective in reducing solution fluorescence.

In one embodiment of the invention, a photon producing agent is excitedwith light of a narrow wavelength bandwidth, such as light filteredthrough a band pass excitation filter or by excitation with a narrowwavelength band or single wavelength of laser light as is known in theart. The emission from the sample is detected using a selected bandwidthof light emitted from the photon producing agent using, for example, anemission band pass filter. Preferably, the photon reducing agent has ahigh extinction coefficient at the excitation and emission wavelengths.

Depending on the sensitivity or reactivity of the reagents used in theassay, such as membrane compartments (such as cells) or targets, it willbe desirable to select photon reducing agents with little or no relevantbiological activity so that the photon reducing agent does not interferewith an assay. Often it will be valuable to test the toxicity andnon-specific binding of the photon reducing agents in the assay toinsure their compatibility with assay components. Preferably, photonreducing agents are not toxic to cells used in a cell based assay withinthe time frame of the assay. Preferably, photon reducing agents do notreact with, or bind to, the targets or other biomolecules in the assayto undesirably alter the biological activity or property being measured.Preferably, photon reducing agents should not cross the (cell) membrane.

Samples can comprise one or more or two or more photon producing agentsand one or more or two or more photon reducing agents. In the case ofmultiple photon producing agents or photon reducing agents, thecharacteristics of the photon producing agents or photon reducing agentscan be selected to have desired characteristics. For example, photonreducing agents can be selected to form combinations that haveabsorption spectra that are broader than the absorption spectra of theindividual photon reducing agents. These combinations of photon reducingagents can be used to reduce the emission of undesired light from one ormore photon producing agents in a sample. Such reduced emission ofundesired light from the sample can be accomplished during theexcitation of and emission from the photon producing agent.

Light emitted from a sample can be detected by any appropriate means fora particular assay format. For example, fluorescence can be detectedusing a fluorometer, which can detect epifluorescence. Samples can beprovided in any appropriate container from which a signal can bedetected, such as vials or wells of a microtiter plate. For microtiterplates, the number of wells in a standard 96-well format footprint canbe between about 6 and about 3,456 wells, preferably between about 96wells and 864 wells, and more preferably between about 288 and about 384wells, more preferably greater than about 384 wells (see, U.S. patentapplication Ser. No. 08/868,018 to Coassin et al., filed Jun. 3, 1997).Preferably, the microtiter plate has wells that have a bottom that hasat least a portion that can pass light of a wavelength used in an assay.Membrane compartments in the sample preferably are in contact (physicalcontact or optical contact) with the bottom of such wells. In thisinstance, optical contact means that the presence of a photon reducingagent, at least a portion of the light emitted from said membranecompartment can pass through the bottom of said well. The solutionvolume containing the sample used is dependent upon the volume capacityof the container used in an assay. Preferably, the sample volume isbetween about 100 nanoliters and about 1 milliliter, preferably betweenabout 0.5 microliters and about 0.5 milliliter, and most preferablybetween about 1 microliters and about 250 microliters or between about 3microliters and about 100 microliters.

The number of membrane compartments, such as cells, in a sample ispreferably between about 10 and about 1,000,000,000 membranecompartments and more preferably between about 100 and about 200,000membrane compartments. When the membrane compartments are cells, thecells are preferably living, and are preferably mammalian cells. Samplescan contain a predetermined number of cells or an unknown number ofcells. Samples can contain cells that are members of a clonal populationor a heterogeneous population. Preferably, the membrane compartmentsform a single layer of membrane compartments in optical contact with anappropriate solid surface such that the emission from a photon producingagent can pass through the appropriate solid surface. However, themembrane compartments may form a plurality of such layers. Membranecompartments in optical contact with a solid surface can be in directcontact with the solid surface, preferably between about 5 Å to aboutthe thickness of a eukaryotic cell in culture, more preferably betweenabout 5 Å to about one-half the thickness of a eukaryotic cell inculture, more preferably between about 10 Å to about the thickness oftwo IgG antibodies placed Fab region to Fab region, more preferablybetween about 15 Å and about the thickness of a lipid bilayer or aboutthe thickness of a cytoplasmic membrane of a eurkaryotic cell inculture.

Cells

Many cells can be used in the invention for cell based assays. Suchcells include, but are not limited to; baby hamster kidney (BHK) cells(ATCC No. CCL10), mouse L cells (ATCC No. CCLI.3), Jurkats (ATCC No. TIB152) and 153 DG44 cells (see, Chasin (1986) Cell. Molec. Genet. 12: 555)human embryonic kidney (HEK) cells (ATCC No. CRL1573), Chinese hamsterovary (CHO) cells (ATCC Nos. CRL9618, CCL61, CRL9096), PC12 cells (ATCCNo. CRL17.21), COS-7 cells (ATCC No. CRL1651) and yeast. Preferred cellsfor heterologous cell surface protein expression are those that can bereadily and efficiently transfected. Preferred cells include Jurkatcells, CHO cells, and HEK 293 cells, such as those described in U.S.Pat. No. 5,024,939 and by Stillman et al. (1985) Mol. Cell. Biol. 5:2051-2060, each of which are incorporated herein by reference.

Targets

One method of the present invention uses targets for identifyingchemicals that are useful for modulating the activity of the target or atarget having similar structural or functional characteristics. Thetarget can be any biological entity, such as a protein, sugar, nucleicacid or lipid. Typically, targets will be proteins such as enzymes orcell surface proteins. Targets can be assayed in either biochemicalassays (targets free of cells) or cell based assays (targets associatedwith a cell).

For example, cells may be loaded with ion or voltage sensitive dyes toreport receptor or ion channel activity, such as calcium channels, orN-methyl-D-aspartate (NMDA) receptors, GABA receptors, kainate/AMPAreceptors, nicotinic acetylcholine receptors, sodium channels, calciumchannels, potassium channels, excitatory amino acid (EAA) receptors, andnicotinic acetylcholine receptors. Assays for determining activity ofsuch receptors can also use agonists and antagonists to use as negativeor positive controls to assess the activity of tested chemicals. Inpreferred embodiments of automated assays for identifying chemicals thathave the capacity to modulate the function of receptors or ion channels(e.g., agonists, antagonists), changes in the level of ions in thecytoplasm or membrane voltage will be monitored using an ion-sensitiveor membrane voltage fluorescent indicator, respectively. Among theion-sensitive indicators and voltage probes that may be employed arethose disclosed in the Molecular Probes 1997 Catalog, hereinincorporated by reference.

Other methods of the present invention concern determining the activityof receptors. Receptor activation can sometimes initiate subsequentintracellular events that release intracellular stores of calcium ionsfor use as a second messenger or the influx of calcium ions into a cell.Activation of some G-protein-coupled receptors stimulates the formationof inositol triphosphate (IP3 a G-protein coupled receptor or tyrosinekinase second messenger) through phospholipase C-mediated hydrolysis ofphosphatidylinositol, Berridge and Irvine (1984), Nature 312: 315-21.IP3 in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels caused by the releaseof calcium ions from intracellular stores can be used to reliablydetermine G-protein-coupled receptor function. Among G-protein-coupledreceptors are muscarinic acetylcholine receptors (mAChR), adrenergicreceptors, serotonin receptors, dopamine receptors, angiotensinreceptors, adenosine receptors, bradykinin receptors, metabotropicexcitatory amino acid receptors, and the like. Cells expressing suchG-protein-coupled receptors may exhibit increased cytoplasmic calciumlevels as a result of contribution from both intracellular stores andvia activation of ion channels. In such instances, it may be desirable,although not necessary, to conduct such assays in calcium-free buffer,optionally supplemented with a chelating agent such as EGTA, todistinguish fluorescence response resulting from calcium release frominternal stores.

Exemplary membrane proteins that may be targets include, but are notlimited to, surface receptors and ion channels. Surface receptorsinclude, but are not limited to, muscarinic receptors, e.g., human M2(GenBank accession #M16404); rat M3 (GenBank accession #M16407), humanM4 (GenBank accession #M16405), human M5 (Bonner, et al., (1988) Neuron1, pp. 403-410); and the like. Neuronal nicotinic acetylcholinereceptors include, e.g., the human α2, α3, and β2, subtypes, the humanα5 subtype (Chini, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:1572-1576), the rat α2 subunit (Wada, et al. (1988) Science 240, pp.330-334), the rat α3 subunit (Boulter, et al. (1986) Nature 319, pp.368-374), the rat α4 subunit (Goldman, et al. (1987) Cell 48, pp.965-973), the rat α5 subunit (Boulter, et al. (1990) I. Biol. Chem. 265,pp. 4472-4482), the chicken α7 subunit (Couturier et al. (1990) Neuron5: 847-856), the rat β2 subunit (Deneris, et al. (1988) Neuron 1, pp.45-54), the rat β3 subunit (Deneris, et al. (1989) J. Biol. Chem. 264,pp. 6268-6272), the rat β4 subunit (Duvoisin, et al. (1989) Neuron 3,pp. 487-496), combinations of the rat α subunits, β subunits and a and psubunits. GABA receptors include, e.g., the bovine n, and p, subunits(Schofield, et al. (1987) Nature 328, pp. 221-227), the bovine n, and a,subunits (Levitan, et al. (1988) Nature 335, pp. 76-79), the γ-subunit(Pritchett, et al. (1989) Nature 338, pp. 582-585), the p, and p,subunits (Ymer, et al. (1989) EMBO J. 8, pp. 1665-1670), the 6 subunit(Shivers, B. D. (1989) Neuron 3, pp. 327-337), and the like. Glutamatereceptors include, e.g., rat GluR1 receptor (Hollman, et al. (1989)Nature 342, pp. 643-648), rat GluR2 and GluR3 receptors (Boulter et al.(1990) Science 249:1033-1037, rat GluR4 receptor (Keinanen et al. (1990)Science 249: 556-560), rat GluR5 receptor (Bettler et al. (1990) Neuron5: 583-595), rat GluR6 receptor (Egebjerg et al. (1991) Nature 351:745-748), rat GluR7 receptor (Bettler et al. (1992) neuron 8:257-265),rat NMDAR1 receptor (Moriyoshi et al. (1991) Nature 354:31-37 andSugihara et al. (1992) Biochem. Biophys. Res. Comm. 185:826-832), mouseNMDA e1 receptor (Meguro et al. (1992) Nature 357: 70-74), rat NMDAR2A,NMDAR2B and NMDAR2C receptors (Monyer et al. (1992) Science 256:1217-1221), rat metabotropic mGluR1 receptor (Houamed et al. (1991)Science 252: 1318-1321), rat metabotropic mGluR2, mGluR3 and mGluR4receptors (Tanabe et al. (1992) Neuron 8:169-179), rat metabotropicmGluR5 receptor (Abe et al. (1992) I. Biol. Chem. 267: 13361-13368), andthe like. Adrenergic receptors include, e.g., human p1 (Frielle, et al.(1987) Proc. Natl. Acad. Sci. 84, pp. 7920-7924), human α2 (Kobilka, etal. (1987) Science 238, pp. 650-656), hamster β2 (Dixon, et al. (1986)Nature 321, pp. 75-79), and the like. Dopamine receptors include, e.g.,human D2 (Stormann, et al. (1990) Molec. Pharm. 37, pp. 1-6), mammaliandopamine D2 receptor (U.S. Pat. No. 5,128,254), rat (Bunzow, et al.(1988) Nature 336, pp. 783-787), and the like. NGF receptors include,e.g., human NGF receptors (Johnson, et al. (1986) Cell 47, pp. 545-554),and the like. Serotonin receptors include, e.g., human 5HT1a (Kobilka,et al. (1987) Nature 329, pp. 75-79), serotonin 5HT1C receptor (U.S.Pat. No. 4,985,352), human 5HT1D (U.S. Pat. No. 5,155,218), rat 5HT2(Julius, et al. (1990) PNAS 87, pp. 928-932), rat 5HT1c (Julius, et al.(1988) Science 241, pp. 558-564), and the like.

Ion channels include, but are not limited to, calcium channels comprisedof the human calcium channel α2 β and/or γ-subunits (see WO89/09834;human neuronal α2 subunit), rabbit skeletal muscle a1 subunit (Tanabe,et al. (1987) Nature 328, pp. 313-E318), rabbit skeletal muscle α2subunit (Ellis, et al. (1988) Science 241, pp. 1661-1664), rabbitskeletal muscle p subunit (Ruth, et al. (1989) Science 245, pp.1115-1118), rabbit skeletal muscle γ subunit (Jay, et al. (1990) Science248, pp. 490-492), and the like. Potassium ion channels include, e.g.,rat brain (BK2) (McKinnon, D. (1989) J. Biol Chem. 264, pp. 9230-8236),mouse brain (BK1) (Tempel, et al. (1988) Nature 332, pp. 837-839), andthe like. Sodium ion channels include, e.g., rat brain I and II (Noda,et al. (1986) Nature 320, pp. 188-192), rat brain III (Kayano, et al.(1988) FEBS Lett. 228, pp. 187-1.94), human II (ATCC No. 59742, 59743and Genomics 5: 204-208 (1989), chloride ion channels (Thiemann, et al.(1992), Nature 356, pp. 57-60 and Paulmichl, et al. (1992) Nature 356,pp. 238-241), and others known or developed in the art.

Intracellular receptors may also be used as targets, such as estrogenreceptors, glucocorticoid receptors, androgen receptors, progesteronereceptors, and mineralocorticoid receptors, in the invention.Transcription factors and kinases can also be used as targets, as wellas plant targets.

Various methods of identifying activity of a chemical with respect to atarget can be applied, including: ion channels (PCT publication WO93/13423), intracellular receptors (PCT publication WO 96/41013), U.S.Pat. Nos. 5,548,063, 5,171,671, 5,274,077, 4,981,784, EP 0 540 065 A1,U.S. Pat. Nos. 5,071,773, and 5,298,429. Fluorescent assays that can beused with the invention include those described in PCT WO 96/3540(Tsien), PCT WO 96/41166 (Tsien) and PCT WO 96/23810 (Tsien). Themethods set forth in PCT WO 96/3540 (Tsien) and PCT WO 96/23810 (Tsien)can also be combined with methods described in U.S. Pat. Nos. 5,401,629and 5,436,128 by Harpold et al. for assays of cell surface receptors andthe cell based intracellular receptor assays referenced herein. All ofthe foregoing references are herein incorporated by reference in theirentirety.

Fluorescence Measurements

When using fluorescent sensors, indicators or probes such as photonproducing agents, it will be recognized that different types offluorescent monitoring systems can be used to practice the invention.Preferably, FACS systems or systems dedicated to high throughputscreening, e.g 96 well or greater microtiter plates or multi-wellplatforms are used to identify compounds such as therapeutic compoundsand to assess the toxicology of such compounds (see U.S. applicationSer. No. 08/858,016 to Styli et al, filed May 16, 1997). Such highthroughput screening systems can comprise, for example:

-   -   a) a storage and retrieval module for storing a plurality of        chemicals in solution in addressable chemical wells, a chemical        well retriever, and having programmable selection and retrieval        of said addressable chemical wells, and having a storage        capacity for at least 100,000 said addressable wells,        -   wherein at least one of said addressable wells comprises a            photon reducing agent,    -   b) a sample distribution module comprising a liquid handler to        aspirate or dispense solutions from selected said addressable        chemical wells, said chemical distribution module having        programmable selection of, and aspiration from, said selected        addressable chemical wells and programmable dispensation into        selected addressable sample wells, and said liquid handler can        dispense into arrays of addressable wells with different        densities of addressable wells per centimeter squared,    -   c) a sample transporter to transport said selected addressable        chemical wells to said sample distribution module and optionally        having programmable control of transport of said selected        addressable chemical wells,    -   d) a reaction module comprising either a reagent dispenser to        dispense reagents into said selected addressable sample wells or        a fluorescent detector to detect chemical reactions ins said        selected addressable sample wells, and    -   e) a data processing and integration module,        -   wherein said storage and retrieval module, said sample            distribution module, and said reaction module are integrated            and programmably controlled by said data processing and            integration module; and said storage and retrieval module,            said sample distribution module, said sample transporter,            said reaction module and said data processing and            integration module are operably linked to facilitate rapid            processing of said addressable sample wells.

Multi-well platforms useful in the present invention can have betweenabout 6 and about 5,000 wells, preferably between about 96 and about4,000 wells, most preferably in multiples of 96 (see U.S. patentapplication Ser. No. 08/867,567, filed Jun. 2, 1997; U.S. patentapplication Ser. No. 08/868,018, filed Jun. 3, 1997; U.S. patentapplication Ser. No. 08/867,584, filed Jun. 2, 1997; U.S. patentapplication Ser. No. 08/868,049, filed Jun. 3, 1997; U.S. patentapplication Ser. No. 09/030,578, filed Feb. 24, 1998; and U.S. patentapplication Ser. No. 09/028,283, filed Feb. 24, 1998). Methods ofperforming assays on fluorescent materials are well known in the art andare described in, e.g., Lakowicz, J. R., Principles of FluorescenceSpectroscopy, New York:Plenum Press (1983); Herman, B., Resonance energytransfer microscopy, in: Fluorescence Microscopy of Living Cells inCulture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. &Wang, Y.-L., San Diego,: Academic Press (1989), pp. 219-243:Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin-CummingsPublishing Co. Inc. (1978), pp. 296-361.

The present invention can be used to increase the signal-to-noise ratioin fluorescence activated cell sorting (FACs). In this aspect of theinvention, the membrane compartment comprises at least one photonproducing agent and the surrounding solution exhibits unwanted opticalbackground (such as fluorescence) from, for example, at least one photonproducing agent. In this embodiment of the invention, the sample volumeis preferably a small droplet comprising the membrane compartment, suchas are useful in FACs analysis. The optical path length through thedroplet is preferably only a few micrometers, so that the reduction ofthe excitation light or absorption of the emitted fluorescence byabsorptive filtering in the droplet is small. Significant reduction ofunwanted solution fluorescence from the droplet can be achieved undersuch conditions when the photon reducing agent in the droplet caninteract with the excited state of the molecule which is the source ofthe unwanted solution fluorescence. Interactions that give rise to suchbeneficial reduction in unwanted solution fluorescence include, but arenot limited to, fluorescence resonance energy transfer, collisionquenching, ground state dark complex formation, paramagnetic enhancementof intersystem crossing, Dexter exchange coupling, photo-inducedelectron transfer. The common property of these interactions is thatthey occur over molecular distances of less than about 20 nm, andcomprise a form of energy transfer other than simple absorption due toinner filtering. The particular conditions for this aspect of theinvention can be determined using routine experimentation using themethods of the present invention.

Fluorescence in a sample can be measured using a fluorimeter. Ingeneral, excitation radiation from an excitation source having a firstwavelength passes through excitation optics. The excitation optics allowthe excitation radiation to excite the sample. In response, fluorescentprobes in the sample emit radiation that has a wavelength that isdifferent from the excitation wavelength. Collection optics then collectthe emission from the sample. The device can include a temperaturecontroller to maintain the sample at a specific temperature while it isbeing measured. According to one embodiment, a multi-axis translationstage moves a microtiter plate holding a plurality of samples in orderto position different wells to be exposed. The multi-axis translationstage, temperature controller, auto-focusing feature, and electronicsassociated with imaging and data collection can be managed by anappropriately programmed digital computer. The computer also cantransform the data collected during the assay into another format forpresentation.

Preferably, fluorescence resonance energy transfer (FRET), can be usedas a way of monitoring activity inside a cell, such as with the reportergene system described in Tsien et al (PCT WO96/30540). The degree ofFRET can be determined by any appropriate spectral or fluorescencelifetime characteristic of the excited construct. For example, thedegree of FRET can be measured by determining the intensity of thefluorescent signal from the donor, the intensity of fluorescent signalfrom the acceptor, the ratio of the fluorescence amplitudes near theacceptor's emission maxima to the fluorescence amplitudes near thedonor's emission maximum, or the excited state lifetime of the donor.For example, cleavage of the linker increases the intensity offluorescence from the donor, decreases the intensity of fluorescencefrom the acceptor, increases the ratio of fluorescence amplitudes fromthe donor to that from the acceptor, and increases the excited statelifetime of the donor.

As would be readily appreciated by those skilled in the art, theefficiency of fluorescence resonance energy transfer depends on thefluorescence quantum yield of the donor fluorophore, the orientation ofthe fluorophore, the donor-acceptor distance, and the overlap integralof donor fluorescence emission and acceptor absorption. The energytransfer is most efficient when a donor fluorophore with highfluorescence quantum yield (preferably, one approaching 100%) is pairedwith an acceptor with a large extinction coefficient at wavelengthscoinciding with the emission of the donor. The dependence offluorescence energy transfer on the above parameters has been reported(Forster, T. (1948) Ann. Physik 2: 55-75; Lakowicz, J. R., Principles ofFluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B.,Resonance energy transfer microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, Vol 30, ed.Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.219-243; Turro, N. J., Modern Molecular Photochemistry, Menlo Part:Benjamin/Cummings Publishing Co., Inc. (1978), pp. 296-361). Also,tables of spectral overlap integrals are readily available to thoseworking in the field (for example, Berlman, I. B. Energy transferparameters of aromatic compounds, Academic Press, New York and London(1973)). The distance between the donor and acceptor at which FREToccurs with 50% efficiency is termed R₀ and can be calculated from thespectral overlap integrals. For the donor-acceptor pairfluorescein-tetramethyl rhodamine, which is frequently used for distancemeasurement in proteins, this distance R₀ is around 50-70 Å (dosRemedios, C. G. et al. (1987) J. Muscle Research and Cell Motility8:97-117). The distance at which the energy transfer in this pairexceeds 90% is about 45 Å.

Preferably, changes in the degree of FRET are determined as a functionof the change in the ratio of the amount of fluorescence from the donorand acceptor, an analysis process referred to as “ratioing.” Changes inthe absolute amount of substrate, excitation intensity, and turbidity orother background absorbances in the sample at the excitation wavelengthaffect the intensities of fluorescence from both the donor and acceptorapproximately in parallel. Therefore, the ratio of the two emissionintensities is a preferred and more robust measure of cleavage thaneither intensity alone.

The excitation state lifetime of the donor moiety is, likewise,independent of the absolute amount of substrate, excitation intensity,or turbidity or other background absorbances. Its measurement requiresequipment with nanosecond time resolution, except in the special case oftransition metal complexes, such as lanthanide complexes, in which casemicrosecond to millisecond resolution is sufficient.

The ratiometric fluorescent reporter system described herein hassignificant advantages over existing reporters for gene integrationanalysis, as it allows sensitive detection and isolation of bothexpressing and non-expressing single living cells. This assay systemuses a non-toxic, non-polar fluorescent substrate that is easily loadedand then trapped intracellularly. Cleavage of the fluorescent substrateby beta-lactamase yields a fluorescent emission shift as substrate isconverted to product. Because the beta-lactamase reporter readout isratiometric, it is unique among reporter gene assays because it controlsfor variables such as the amount of substrate loaded into individualcells. The stable, easily detected, intracellular readout eliminates theneed for establishing clonal cell lines prior to expression analysis.

Method of Screening Test Chemicals in Fluorescent Assays Using at LeastOne Photon Reducing Agent

The present invention also includes a method of identifying a chemicalwith a biological activity (including toxicological activities). Themethod is performed by contacting a sample with a test chemical, whereinthe sample comprises a target and a photon producing agent. The sampleis also contacted with at least one photon reducing agent and theoptical signal from the photon producing agent is detected. The photonreducing agent may be added before or after the test chemical, asappropriate. The sample can include a membrane compartment in contactwith a solid surface that can pass light. The membrane compartmentincludes at least one photon producing agent that directly or indirectlymonitors the activity of the target. The photon reducing agent is in anaqueous solution that contacts the outer surface of said membranecompartment. Preferably, the photon reducing agent has an absorptionspectra that overlaps with the absorption, emission or excitationspectrum of the photon producing agent or of a second photon producingagent in said aqueous solution. Also, the first photon producing agentcan, in some instances, transfer fluorescence resonance energy to thephoton reducing agent. Alternatively, the second photon producing agentin an aqueous solution can transfer fluorescence resonance energy to thephoton reducing agent. The present invention also includes a therapeuticcompound or composition identified by this method.

A test chemical with a biological activity, such as a therapeutic, canbe identified by contacting a test chemical suspected of having abiological activity with a sample comprising a membrane compartmentcomprising a target, such as a mammalian cell comprising a receptor. Insome assays, the binding of a test chemical with a target can result inthe expression of a reporter gene in the membrane compartment. Thereporter gene can encode a photon producing agent or precursor photonproducing agent, or encode an enzyme that can produce a photon producingagent from an appropriate substrate. If the sample contains a testchemical with a biological activity reported by the reporter gene, thenthe amount of a fluorescent reporter product in the sample, such asinside or outside of the cell, will either increase or decrease relativeto background or control levels. The amount of the fluorescent reporterproduct is measured by exciting the fluorescent reporter product with anappropriate radiation of a first wavelength and measuring the emissionof radiation of a second wavelength emitted from said sample. The amountof emission is compared to background or control levels of emission. Ifthe sample having the test chemical exhibits increased or decreasedemission relative to that of the control or background levels, then acandidate modulator has been identified. The amount of emission isrelated to the amount or potency of the therapeutic in the sample. Suchmethods are described in, for example, Tsien (PCT/US90/04059). Suchmethods identify candidate modulators of biological processes. Thecandidate modulator can be further characterized and monitored forstructure, potency, toxicology, and pharmacology using well knownmethods or those described herein.

The signal-to-noise ratio of such assays can be increased by theaddition of at least one photon reducing agent during the course of suchassays. The addition of at least one photon reducing agent can alsoincrease the sensitivity and precision of such assays.

The structure of a candidate modulator identified by the invention canbe determined or confirmed by methods known in the art, such as massspectroscopy and nuclear magnetic resonance (NMR). For putativechemicals with a biological activity stored for extended periods oftime, the structure, activity, and potency of candidate modulators canbe confirmed.

Depending on the system used to identify candidate modulators, acandidate modulator can have putative pharmacological activity. Forexample, if the candidate modulator is found to inhibit T-cellproliferation (activation) in vitro, then the candidate modulator wouldhave presumptive pharmacological properties as an immunosuppressant oranti-inflammatory (see, Suthanthiran et al., Am. J. Kidney Disease,28:159-172 (1996)). Such nexuses are known in the art for severaldisease states, and more are expected to be discovered over time. Basedon such nexuses, appropriate confirmatory in vitro and in vivo models ofpharmacological activity, as well as toxicology, can be selected. Themethods described herein can also be used to assess pharmacologicalselectivity and specificity, and toxicity.

Bioavailability and Toxicology of Candidate Modulators

Once identified, candidate modulators can be evaluated forbioavailability and toxicological effects using known methods (see, Lu,Basic Toxicology, Fundamentals, Target Organs, and Risk Assessment,Hemisphere Publishing Corp., Washington (1985); U.S. Pat. No. 5,196,313to Culbreth (issued Mar. 23, 1993) and U.S. Pat. No. 5,567,952 to Benet(issued Oct. 22, 1996). For example, toxicology of a candidate modulatorcan be established by determining in vitro toxicity towards a cell line,such as a mammalian i.e. human, cell line. Candidate modulators can betreated with, for example, tissue extracts, such as preparations ofliver, such as microsomal preparations, to determine increased ordecreased toxicological properties of the chemical after beingmetabolized by a whole organism. The results of these types of studiesare often predictive of toxicological properties of chemicals inanimals, such as mammals, including humans.

Such bioavailability and toxicological methods can be performed usingthe methods, preferable using the screening systems of the presentinvention. Such methods include contacting a sample having a target withat least one photon producing agent, at least one photon reducing agent,and a test chemical. An optical signal from said at least one photonproducing agent is detected, wherein said optical signal is related to atoxicological activity. Bioavailability is any known in the art and canbe detected, for example by measuring reporter genes that are activatedduring bioavailability criteria. Toxicological activity is any known inthe art, such as apoptosis, cell lysis, crenation, cell death and thelike. The toxicological activity can be measured using reporter genesthat are activated during toxicological activity or by cell lysis (seeWO 98/13353, published Apr. 2, 1998). Preferred reporter genes produce afluorescent or luminescent translational product (such as, for example,a Green Fluorescent Protein (see, for example, U.S. Pat. No. 5,625,048to Tsien et al., issued Apr. 29, 1998; U.S. Pat. No. 5,777,079 to Tsienet al., issued Jul. 7, 1998; WO 96/23810 to Tsien, published Aug. 8,1996; WO 97/28261, published Aug. 7, 1997; PCT/US97/12410, filed Jul.16, 1997; PCT/US97/14595, filed Aug. 15, 1997)) or a translationalproduct that can produce a fluorescent or luminescent product (such as,for example, beta-lactamase (see, for example, U.S. Pat. No. 5,741,657to Tsien, issued Apr. 21, 1998, and WO 96/30540, published Oct. 3,1996)), such as an enzymatic degradation product. Cell lysis can bedetected in the present invention as a reduction in a fluorescencesignal from at least one photon producing agent within a cell in thepresence of at least one photon reducing agent. Such toxicologicaldeterminations can be made using prokaryotic or eukaryotic cells,optionally using toxicological profiling, such as described inPCT/US94/00583, filed Jan. 21, 1994, German Patent No 69406772.5-08,issued Nov. 25, 1997; EPC 0680517, issued Nov. 12, 1994; U.S. Pat. No.5,589,337, issued Dec. 31, 1996; EPO 651825, issued Jan. 14, 1998; andU.S. Pat. No. 5,585,232, issued Dec. 17, 1996)

Alternatively, or in addition to these in vitro studies, thebioavailability and toxicological properties of a candidate modulator inan animal model, such as mice, rats, rabbits, or monkeys, can bedetermined using established methods (see, Lu, supra (1985): andCreasey, Drug Disposition in Humans. The Basis of Clinical Pharmacology,Oxford University Press, Oxford (1979), Osweiler, Toxicology, Williamsand Wilkins, Baltimore, Md. (1995), Yang, Toxicology of ChemicalMixtures; Case Studies, Mechanisms, and Novel Approaches, AcademicPress, Inc., San Diego, Calif. (1994), Burrell et al., Toxicology of theImmune System; A Human Approach, Van Nostrand Reinhold, Co. (1997),Niesink et al., Toxicology; Principles and Applications, CRC Press. BocaRaton, Fla. (1996)). Depending on the toxicity, target organ, tissue,locus, and presumptive mechanism of the candidate modulator, the skilledartisan would not be burdened to determine appropriate doses, LD₅₀values, routes of administration; and regimes that would be appropriateto determine the toxicological properties of the candidate modulator. Inaddition to animal models, human clinical trials can be performedfollowing established procedures, such as those set forth by the UnitedStates Food and Drug Administration (USFDA) or equivalents of othergovernments. These toxicity studies provide the basis for determiningthe efficacy of a candidate modulator in vivo.

Efficacy of Candidate Modulators

Efficacy of a candidate modulator can be established using several artrecognized methods, such as in vitro methods, animal models, or humanclinical trials (see, Creasey, supra (1979)). Recognized in vitro modelsexist for several diseases or conditions. For example, the ability of achemical to extend the life-span of HIV-infected cells in vitro isrecognized as an acceptable model to identify chemicals expected to beefficacious to treat HIV infection or AIDS (see, Daluge et al.,Antimicro. Agents Chemother. 41:1082-1093 (1995)). Furthermore, theability of cyclosporin A (CsA) to prevent proliferation of T-cells invitro has been established as an acceptable model to identify chemicalsexpected to be efficacious as immunosuppressants (see, Suthanthiran etal., supra, (1996)). For nearly every class of therapeutic, disease, orcondition, an acceptable in vitro or animal model is available. Suchmodels exist, for example, for gastro-intestinal disorders, cancers,cardiology, neurobiology, and immunology. In addition, these in vitromethods can use tissue extracts, such as preparations of liver, such asmicrosomal preparations, to provide a reliable indication of the effectsof metabolism on the candidate modulator. Similarly, acceptable animalmodels may be used to establish efficacy of chemicals to treat variousdiseases or conditions. For example, the rabbit knee is an acceptedmodel for testing chemicals for efficacy in treating arthritis (see,Shaw and Lacy, J. Bone Joint Surg. (Br) 55:197-205 (1973)).Hydrocortisone, which is approved for use in humans to treat arthritis,is efficacious in this model which confirms the validity of this model(see, McDonough, Phys. Ther. 62:835-839 (1982)). When choosing anappropriate model to determine efficacy of a candidate modulator, theskilled artisan can be guided by the state of the art to choose anappropriate model, dose, and route of administration, regime, andendpoint and as such would not be unduly burdened

In addition to animal models, human clinical trials can be used todetermine the efficacy of a candidate modulator in humans. The USFDA, orequivalent governmental agencies, have established procedures for suchstudies (see www.fda.gov).

Selectivity of Candidate Modulators

The in vitro and in vivo methods described above also establish theselectivity of a candidate modulator. It is recognized that chemicalscan modulate a wide variety of biological processes or be selective.Panels of cells based on the present invention can be used to determinethe specificity of the candidate modulator. Selectivity is evident, forexample, in the field of chemotherapy, where the selectivity of achemical to be toxic towards cancerous cells, but not towardsnon-cancerous cells, is obviously desirable. Selective modulators arepreferable because they have fewer side effects in the clinical setting.The selectivity of a candidate modulator can be established in vitro bytesting the toxicity and effect of a candidate modulator on a pluralityof cell lines that exhibit a variety of cellular pathways andsensitivities. The data obtained from these in vitro toxicity studiescan be extended animal model studies, including human clinical trials,to determine toxicity, efficacy, and selectivity of the candidatemodulator.

Identified Compositions

The invention includes compositions such as novel chemicals, andtherapeutics identified as having activity by the operation of methods,systems or components described herein. Novel chemicals, as used herein,do not include chemicals already publicly known in the art as of thefiling date of this application. Typically, a chemical would beidentified as having activity from using the invention and then itsstructure revealed from a proprietary database of chemical structures ordetermined using analytical techniques such as mass spectroscopy.

One embodiment of the invention is a chemical with useful activity,comprising a chemical identified by the method described above. Suchcompositions include small organic molecules, nucleic acids, peptidesand other molecules readily synthesized by techniques available in theart and developed in the future. For example, the followingcombinatorial compounds are suitable for screening: peptoids (PCTPublication No. WO 91/19735, 26 Dec. 1991), encoded peptides (PCTPublication No. WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCTPublication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No.5,288,514), diversomeres such as hydantoins, benzodiazepines anddipeptides (Hobbs DeWitt, S. et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer.Chem. Soc. 114: 6568 (1992)), nonpeptidal peptidomimetics with aBeta-D-Glucose scaffolding (Hirschmann, R. et al., J. Amer. Chem. Soc.114: 9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen, C. et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho, C. Y. et al., Science 261: 1303 (1993)), and/orpeptidyl phosphonates (Campbell, D. A. et al., J. Org. Chem. 59: 658(1994)). See, generally, Gordon, E. M. et al., J. Med Chem. 37: 1385(1994). The contents of all of the aforementioned publications areincorporated herein by reference.

The present invention also encompasses the identified chemicals andtheir respective compositions, typically in a pharmaceutical compositionof the present invention that comprise a pharmaceutically acceptablecarrier prepared for storage and subsequent administration, which havethe pharmaceutically effective amount of the products disclosed above ina pharmaceutically acceptable carrier or diluent. Acceptable carriers ordiluents for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington's Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives,stabilizers, dyes and even flavoring agents may be provided in thepharmaceutical composition. For example, sodium benzoate, sorbic acidand esters of p-hydroxybenzoic acid may be added as preservatives. Inaddition, antioxidants and suspending agents may be used.

The compositions of the present invention may be formulated and used astablets, capsules or elixirs for oral administration; suppositories forrectal administration; sterile solutions, suspensions for injectableadministration; and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations(e.g., liposomes), may be utilized.

The pharmaceutically effective amount of the composition required as adose will depend on the route of administration, the type of animalbeing treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize.

In practicing the methods of the invention, the products or compositionscan be used alone or in combination with one another, or in combinationwith other therapeutic or diagnostic agents. These products can beutilized in vivo, ordinarily in a mammal, preferably in a human, or invitro. In employing them in vivo, the products or compositions can beadministered to the mammal in a variety of ways, including parenterally,intravenously, subcutaneously, intramuscularly, colonically, rectally,nasally or intraperitoneally, employing a variety of dosage forms. Suchmethods may also be applied to testing chemical activity in vivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage for the products of the present invention can range broadlydepending upon the desired affects and the therapeutic indication.Typically, dosages may be between about 10 kg/kg and 100 mg/kg bodyweight, preferably between about 100 μg/kg and 10 mg/kg body weight.Administration is preferably oral on a daily basis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g., Fingl et al., in The Pharmacological Basis of Therapeutics, 1975).It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theinvention into dosages suitable for systemic administration is withinthe scope of the invention. With proper choice of carrier and suitablemanufacturing practice, the compositions of the present invention, inparticular, those formulated as solutions, may be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the invention to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules maybe directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping, or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dye-stuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. Such formulations can be madeusing methods known in the art (see, for example, U.S. Pat. No.5,733,888 (injectable compositions); U.S. Pat. No. 5,726,181 (poorlywater soluble compounds); U.S. Pat. No. 5,707,641 (therapeuticallyactive proteins or peptides); U.S. Pat. No. 5,667,809 (lipophilicagents); U.S. Pat. No. 5,576,012 (solubilizing polymeric agents); U.S.Pat. No. 5,707,615 (anti-viral formulations); U.S. Pat. No. 5,683,676(particulate medicaments); U.S. Pat. No. 5,654,286 (topicalformulations); U.S. Pat. No. 5,688,529 (oral suspensions); U.S. Pat. No.5,445,829 (extended release formulations); U.S. Pat. No. 5,653,987(liquid formulations); U.S. Pat. No. 5,641,515 (controlled releaseformulations) and U.S. Pat. No. 5,601,845 (spheroid formulations).

EXAMPLES

The structure of CCF2/AM used in the experiments described herein is:

Example 1 Reduction of Solution Fluorescence

To investigate the ability of a photon reducing agent to reducefluorescence of a solution, emission of a fluorophore from a sample wasmonitored in the presence and absence of a photon reducing agent. Thefollowing experiments show that photon reducing agents can be used toreduce solution based fluorescence from a fluorophore.

Fluorescence from a solution of a blue fluorescent dye,6-chloro-7-hydroxy coumarin 3-carboxylate, was determined in thepresence and absence of a 1 mM phenol red as the photon reducing agent.A solution of 0.5 μM 6-chloro-7-hydroxy coumarin-3-carboxylate insolvent containing 50% (vol/vol) aqueous 39 mM phosphate buffer pH 7.5and 50% (vol/vol) methanol was prepared. After taking a front facefluorescence spectrum of this solution on a Spex Fluorolog 2, 1%(vol/vol) of a 100 mM aqueous Phenol Red stock solution was added andanother spectrum taken. Front face fluorescence refers to exciting thesample at a right angle to the sample surface and collecting emittedlight (for example, from the excited region or emission region) at 12.5degrees from such angle of excitation.

FIG. 2 shows these spectra. Addition of phenol red reduced front facefluorescence 70 fold. The photon reducing agent, phenol red,substantially reduced the solution based fluorescence signal. Althoughthe inventors do not wish to be bound by any proposed mechanism, at 50%(vol/vol), the organic solvent (methanol) in the solution may preventassociation of the dye molecules in solution. This is consistent withthe explanation that fluorescence reduction does not requireground-state fluorescence quenching due to dye stacking. This result isconsistent with a photon reducing agent decreasing fluorescence of thefluorophore by absorbing the excitation or emission light to or from thefluorophore or by accepting the energy of the excited singlet state ofthe fluorophore (a state that gives rise to the fluorescence) by along-range energy transfer mechanism, such as fluorescence resonanceenergy transfer.

Example 2 Reduction of Solution Fluorescence is not NecessarilyAssociated with Stacking

To further investigate the ability of a photon reducing agent to reducefluorescence of a solution, fluorescence of a dye in the presence of aphoton reducing agent was monitored in the presence and absence ofmethanol. The following experiment shows that a photon reducing agent,phenol red, can be used to reduce fluorescence from a fluorophore inaqueous solution without dye stacking.

The fluorescence from a fluorophore in the presence of a photon reducingagent (phenol red) was determined in both aqueous buffer and aqueousbuffer containing 50% (vol/vol) methanol. A 10 μM solution of6-chloro-7-hydroxy coumarin-3-carboxylate was prepared in 39 mMphosphate buffer pH 7.5 and in a 1:1 mixture of same phosphate bufferand methanol. 1% (vol/vol) of a 100 mM aqueous Phenol Red stock solutionwas added to the solutions.

FIG. 3 shows front-face fluorescence spectra of these solutions wereobtained on a Spex Fluorolog 2. The presence of methanol increasedfluorescence compared to an all aqueous buffer. These results furtherconfirm that the fluorescence decrease observed in Example 1 was notentirely due to dye stacking. Note the relative fluorescence in Example2 is comparable or less than the fluorescence of coumarin/phenol redsample of Example 1.

The increased fluorescence in the presence of methanol in the buffer isconsistent with finding that 50% methanol buffer increased6-chloro-7-hydroxy coumarin-3-carboxylate fluorescence in the absence ofa photon reducing agent by about 30 percent. The increased fluorescencein the presence of methanol in the buffer is also consistent withfinding that the 50% methanol buffer decreased the absorbance of thephoton reducing agent phenol red at the emission wavelength of thecoumarin by about 20 percent. Under these conditions, stacking of thedye-based photon reducing agent and the photon producing agent does notcontribute significantly to fluorescence-reduction effect of dye-basedphoton reducing agents. Dye stacking is especially unlikely when thefluorophore is very water soluble and small, such as 6-chloro-7-hydroxycoumarin-3-carboxylate.

Example 3 Test of Reduction of Solution Fluorescence Using Non-DyeQuenchers and Particulates

To investigate the ability of a candidate photon reducing agent toreduce fluorescence of a solution, fluorescence of a fluorophore in thepresence of a candidate photon reducing agent was monitored as afunction of photon reducing agent concentration. The candidate photonreducing agents used were non-dye molecules and particulates. Thefollowing experiments show that non-dye molecules and particulates canbe used as photon reducing agents to reduce fluorescence from afluorophore in aqueous buffer.

Signals from fluorescent dye solutions containing no photon reducingagents or photon reducing agents, such as Schilling Red (water,propylene glycol, FD&C Red No. 40 (Allura red), FD&C Red No. 3 andpropyparaben (McCormick & Co., Inc. Hunt Valley, Md.) and phenol red,were compared to solutions containing candidate photon reducing agents,non-dye molecules (diatrizoic acid and Tris(2-amino ethyl)amine) andparticulate ink (Higgins ink). In a 96 well microtiter plate, two foldserial dilutions of aqueous 0.5 M diatrizoic acid and 1 Mtris(2-aminoethyl)amine (adjusted to pH 7.5 with hydrochloric acid), 100mM phenol red, Schilling Red food dye and Higgins ink were prepared inthe presence of 10 μM 6-chloro-7-hydroxy coumarin-3-carboxylate in the39 mM phosphate buffer (pH 7.5). The well volume was 100 μl. A two-foldserial dilution of 10 μM 6-chloro-7-hydroxy coumarin-3-carboxylate inphosphate buffer pH 7.5 was prepared for comparison. A linear signal wasshown over the range of coumarin concentrations tested. The fluorescenceemission intensity of the samples was measured on a Cytofluor microtiterplate fluorimeter. The samples were excited with 395 nm light andfluorescence emission measured at 460 nm. Data for the Higgins ink andSchilling Red solutions were normalized by absorbance at 395 nm and 460nm to the phenol red solution because their concentrations were eitherunknown (Schilling Red) or not defined (Higgins ink).

FIG. 4 graphs the concentration of the candidate photon reducing agentagainst the residual coumarin fluorescence, which shows the dependenceof sample fluorescence on candidate photon reducing agent concentration.Efficient reduction of fluorescence occurs at non-dye concentrationsgreater than 0.5 M, while the particulate Higgins ink and Schilling Redwere similarly effective to phenol red. This result demonstrates thatphoton reducing agents that act as only as collisional quenchers(diatrizoic acid and tris(2-aminoethyl)amine) will typically requireconcentrations higher than 100 mM, which could contribute to ionicstrength effects in potential assays. These results also demonstratethat a photon reducing agent consisting of light-absorbing (or lightscattering) particulate matter, such as an ink, can effectively reducesolution fluorescence.

Example 4 Test of Reduction of Solution Fluorescence Using Dye-BasedPhoton Reducing Agents with Absorbance Spectra Sufficiently Overlappingwith the Emission or Excitation Spectrum of the Photon Producing Agent

To investigate the ability of a candidate dye-based photon reducingagent to reduce fluorescence of a solution, fluorescence of afluorophore in the presence of a candidate photon reducing agent wasmonitored as a function of the photon reducing agent concentration. Thecandidate photon reducing agents were dye molecules with differentabsorption spectra compared to three different fluorophores. Thefollowing experiments demonstrate that dye based photon reducing agentsare most effective in reducing solution based fluorescence when theirabsorption maxima significantly overlaps the excitation and emissionspectra of the fluorophore.

The efficiency with which water soluble dyes (photon reducing agents) ofdifferent colors were able to reduce fluorescence from fluorophoresolutions of 7-hydroxycoumarin, CCF2, fluorescein and rhodamine B wasstudied. A mixture of dye photon reducing agents with high extinctionover the range from 380-555 nm (named Tararaf) was also studied.

TABLE 1 Absorption maxima of dyes Naphthol Yellow: 428 (392) nmTartrazine: 425 nm Phenol Red: 557 (423) nm Acid Red 37: 513 nm AcidFuchsin: 546 nm Trypan Blue: 607 nm Patent Blue: 635 nm Tararaf: 441(513) nm

(Tararaf contains Tartrazine, Acid Red 37 and Acid Fuchsin in a molarratio of 5:6:4)

TABLE 2 Excitation and emission wavelength used in the study offluorophores 6-Chloro-7-hydroxy- ex. 395 nm, em. 460 nmcoumarin-3-carboxylate: CCF2: ex. 395 nm, em. 530 nm Fluorescein: ex.485 nm, em. 530 nm Rhodamine B: ex. 530 nm, em. 595 nm

The dye photon reducing agents were made 20 mM in 39 mM phosphate bufferpH 7.43 containing 10 μM of fluorescent dye. The Tararaf mixturesconcentration was adjusted for its component Tartrazine to be 20 mM. Ina 96-well fluorescence micro titer plate ten two-fold serial dilutionsof these dyes were prepared with buffer containing 10 μM fluorescentdye. The fluorescence of the solutions in the wells was measured using amicrotiter fluorimeter. The values were background subtracted anddivided by the value obtained for 10 μM fluorescent dye in the absenceof photon reducing agent. The values so obtained were termed residualfluorescence.

FIG. 5 shows 6-chloro-7-hydroxycoumarin-3-carboxylate solutionfluorescence as a function of colored photon reducing agentconcentration. Single yellow dyes that absorb coumarin excitation andemission light reduced fluorescence at lower concentrations better thansingle red or blue dyes. Tararaf also effectively reduced solutionfluorescence. The absorbance spectra of the components of Tararafsignificantly overlap with the excitation and emission spectra of thisfluorophore.

FIG. 6 shows fluorescein solution fluorescence as a function of coloredphoton reducing agent concentration. Single yellow and red dyes thatabsorb in the excitation and/or emission spectra of fluorescein reducedsolution fluorescence at lower concentrations better than blue dyes thatabsorb predominantly outside that range of wavelengths. Tararaf alsoeffectively reduced solution fluorescence. The absorbance spectra of thecomponents of Tararaf significantly overlap with the excitation andemission spectra of this fluorophore.

FIG. 7 shows rhodamine solution fluorescence as a function of coloredphoton reducing agent concentration. Red dyes that absorb in excitationspectrum of rhodamine and blue dyes that absorb in the emission spectrumof rhodamine reduced solution fluorescence at lower concentrations morethan yellow dyes that absorb outside that range of wavelengths. Tararafeffectively reduced solution fluorescence. The absorbance spectra of thecomponents of Tararaf significantly overlap with the excitation spectrumof this fluorophore.

FIG. 8 shows residual CCF2 solution fluorescence as a function ofcolored photon reducing agent concentration. Single yellow and red dyesthat absorb in the excitation and/or emission spectra of CCF2 reducedfluorescence at lower concentrations than blue dyes that absorb outsidethat range. Tararaf also effectively reduced solution fluorescence. Theabsorbance spectra of the components of Tararaf significantly overlapwith the excitation and emission spectra of this fluorophore.

These experiments demonstrate that dye-based photon reducing agents aremost effective in reducing solution based fluorescence when theirabsorption maxima lie in the spectral range of the excitation and/oremission of the fluorophore.

Example 5 Test of Reduction of Solution Fluorescence Using Non-Dye BasedPhoton Reducing Agents that Electronically Interact with the PhotonProducing Agent

To investigate the ability of a candidate transition metal based ortransition metal complex based photon reducing agents to reducefluorescence emitted from a solution, fluorescence of a fluorophore inthe presence of a candidate photon reducing agent was monitored as afunction of photon reducing agent concentration. The candidate photonreducing agents used were ions that can potentially electronicallyinteract with a fluorophore. The following experiments demonstrate thatnon-dye based photon reducing agents that are transition metal based ortransition metal complexes can be easily tested and selected to reducesolution based fluorescence of a particular fluorophore. The followingexperiments demonstrate that salts of transition metals and theircomplexes can act as photon reducing agents of specific fluorophores.

Fluorescence was measured with a microtiter plate fluorimeter withexcitation at 395 nm and emission at 460 nm for the coumarinfluorophore, excitation 485 nm and emission at 530 nm for fluoresceinand excitation 530 nm and emission at 590 nm for rhodamine B. 500 mMsolutions of potassium ferrocyanide (II), potassium ferricyanide (III),nickel (II) chloride and copper (II) sulfate were prepared in water. 800μl of each stock solution was diluted with 190 μl 50 mM K-MOPS pH 7.15and 10 μl 1 mM fluorophore solution to a final 400 mM (stock solution).Two-fold serial dilutions of these stock solutions into 10 μMfluorophore containing K-MOPS buffer were prepared in 96 well black(clear bottom) Costar plates. As in Example 4, the measured values werebackground subtracted and normalized to values obtained from fluorophorein absence of photon reducing agents (see FIG. 9).

These experiments demonstrated that fluorescence from coumarinfluorophores can be reduced by iron (III) and nickel (II) salts in thelow millimolar range. Other ions fluorophore combinations demonstratedan affect at higher concentrations (approximately 10 mM and above).

Example 6 Test of Reduction of Solution Fluorescence as S Function ofPath Length

To further investigate the ability of photon reducing agents to reducefluorescence emitted from a solution, fluorescence emitted from asolution containing a fluorophore in the presence of a photon reducingagent was monitored as a function sample thickness. The followingexperiments surprisingly demonstrate that dye-based photon reducingagents reduce solution fluorescence of a fluorophore at shorttransmission distances.

A photon reducing agent, phenol red, was tested with a fluorophore,coumarin, as a function of sample thickness (path length). Theexperiment was conducted using a microscope equipped with epifluorescence. The liquid samples were drawn into low fluorescencecapillary tubes of fixed path length (Vitro Dynamics, Rockaway N.J.) asindicated in the graphs. The following samples were evaluated:

-   -   1) 10 μM coumarin (diamonds)    -   2) 10 μM coumarin+1 mM phenol red (squares)    -   3) 10 μM coumarin+5% (vol/vol) Schilling Red (triangles).

The samples were excited using a 405/20 filter via a 425 dichroicreflector through a 20× objective (Zeiss 20×Fluar). Emitted light wasfiltered through a 460/50 filter and detected by an intensified CCDcamera (Stanford Photonics, Menlo Park, Calif.). The detector output wasconverted to a 512×512 pixel eight-bit digital image. The data reflectthe average intensity within a 20×20 pixel area within the capillary.The background intensity of the field was subtracted from all values.

FIG. 10A shows the raw data for this experiment. Coumarin fluorescencewas significantly attenuated by the presence of the phenol red. Longerpaths are also increasingly attenuated.

FIG. 10B shows the percentage of coumarin fluorescence observed as afunction of path length for each of the dyes tested. The decrease offluorescence at short pathlengths is not consistent with a filteringaffect of the photon reducing agent. At short pathlengths and lowconcentrations of a photon reducing agent there is not a sufficientnumber of photon reducing agent molecules to filter out light.

FIG. 10C shows the calculated decrease in coumarin fluorescence based onfiltering affects. A Beer-Lambert relationship was used to model theexpected decrease in fluorescence due to the effect of filtering lighteither by decreasing the amount of light available for excitation of thefluorophore or the amount of light emitted by the fluorophore.

These results demonstrate that photon reducing agents can be effectivein reducing solution fluorescence in shallow samples, such as low volumeassay samples. This is a surprising result because the amount of dyethat is located in the space between the fluorophore and detector inthis instance is quite small. This effect is consistent withdeactivation of the excited fluorophore by fluorescence resonance energytransfer (FRET) to the photon reducing agents. The average distancebetween molecules in millimolar solutions of photon reducing agents isless than 100 Å. At such short distances FRET has been shown to be veryefficient means of quenching fluorophore fluorescence. Although dyebased photon reducing agents can reduce solution fluorescence atrelatively short pathlengths, such photon reducing agents also allownearly complete transmission (greater than about 80%) through shorterpath lengths (e.g., less than 15 μm), which is appropriate formonitoring most mammalian cells.

Example 7 Photon Reducing Agents Reduce Undesired Fluorescence in CellBased Assays

To investigate the ability of photon reducing agents to reduce undesiredfluorescence of a cell-based assay, fluorescence of a fluorophore in thepresence of a photon reducing agent was monitored using mammalian cells.The following experiments surprisingly demonstrate that photon reducingagents reduce solution based fluorescence of a fluorophore in cell-basedassays.

The fluorescence readout of CCF2 (a substrate for beta-lactamase) in thepresence and in the absence of the photon reducing agents was measured.The derivative CCF2/AM, as described in PCT publication WO96/30540(Tsien), is a vital dye that diffuses into cells and is trapped byliving cells. Cells having esterase activity that cleaves ester groupson the CCF2/AM molecules, which results in a negatively charged moleculeCCF2 that is trapped inside the cell. Trapped dye appears as greenfluorescence inside of living cells devoid of beta-lactamase. Cellsexpressing beta-lactamase show blue fluorescence because the product ofthe beta-lactamase cleavage of CCF2 has blue fluorescence. CCF2 wasincubated with Jurkat cells as previously described (see WO96/30540).These cells were not attached to the microtiter plates but are allowedto settle in the plate wells.

In these experiments, two sets of loading conditions (5 uM CCF2/AMlot#003 and 10 uM CCF2/AM lot#003) and two types of photon reducingagents (5% v/v Schilling Red Food Dye and 0.660 mM (final concentration)phenol red) were used. The presence of photon reducing agents increasedthe signal to noise ratio of the assay at least 200 to 300 percentcompared to the absence of a photon reducing agent. Schilling Red FoodDye can vary from batch to batch. Thus, it is important to test eachbatch before using it in a large series of experiments or cell-basedscreens. In such cell-based assays solution fluorescence is typicallyfrom a fluorophore (such as CCF2/AM or its hydrolysis products) in thecell culture medium that baths the cells.

Beta-lactamase activity is preferably assessed by addition of ⅙^(th)volume CCF2/AM aqueous loading solution containing 6 μM CCF2/AM, 24%PEG-400, 6.2% DMSO, 0.6% Pluronic F127, 7.2 mM Tartrazine, 7.2 mM AcidRed 40 to microtiter wells at room temperature. After 30 min incubation,the fluorescence from the wells is read on a microtiter platefluorimeter with excitation at 395/20 nm and emission at 460/40 nm and530/30 nm. The raw fluorescence emission values were corrected for thesignal from wells devoid of cells. Then, the corrected signal from theblue channel (460/40 nm) was divided by the signal from the greenchannel (530/30 nm). This type of analysis is referred to as ratioing.With the gain settings used for this experiment, a population of greaterthan 95% blue fluorescent cells (>95% cells expressing beta-lactamase)will give a ratio of greater than 3.0 and a population of entirely greenfluorescent cells (no cell expressing any beta-lactamase) will give aratio of about 0.1-0.2.

A comparison to prior art methods to reduce background fluorescence incell-based assays was made. In the “washed assay” format, cells arestimulated to express beta-lactamase, washed, loaded with CCF2/AM,washed with CCF2/AM free media, and then plated out into micro-titerplates for fluorescence readout. Such a protocol with wash steps canwork; however, the protocol has serious limitations and drawbacks forscreening, high-throughput manipulations, and miniaturization. Thewashed format was compared with unwashed cells in the absence andpresence of a photon reducing agent (Red Food Dye) at differentconcentrations ranging from 0 to 1.064 mM final using a fluorescencereadout from a microtiter plate reader described herein. Photon reducingagents used in conjunction with the CCF2/AM substrate ester is oftenreferred to as the “Enhanced Substrate System or ESS.”

FIG. 11 shows that photon reducing agents reduce fluorescence inunwashed cells and yields signals comparable to signals from washedcells. Photon reducing agents also provide much better signals than whenno photon reducing agent is present. All data points are background(buffer plus ESS plus CCF2/AM (no cells)) subtracted. Data is typicallyexpressed as the ratio of ratio. The first ratio is the ratio offluorescence values at the two indicated emission wavelengths (460nm/530 nm) for each experimental data point. The second ratio is theratio of first ratio for the two experimental condition of cellsconstitutively expressing beta-lactamase and wildtype cells (CMVcells/wildtype cells).

Example 8 Testing of Dye-Based Photon Reducing Agents for Cytotoxicityin Cell Based Assays

To investigate the ability of candidate dye-based photon reducing agentsto reduce undesired fluorescence in a cell based assay, cytotoxicity inthe presence of a candidate photon reducing agent was monitored as afunction of the photon reducing agent concentration. The candidatephoton reducing agents were selected from a number of dyes based ontheir absorbance spectra and use with living systems. The followingexperiments demonstrate that dye-based photon reducing agents can beeasily tested and selected for their compatibility with cell-basedassays.

From an initial list of 50 dye compounds, the following dyes wereselected and tested with mammalian cells: Bromophenol Blue, ChlorophenolRed, Tartrazine, Phenol Red, Naphthol Yellow, Chromotrope F8,Chromazurol S, Patent Blue, Chromotrope 2R, Quinoline Yellow, AcidFuchsin, Erythrosin, Acid Red 37, and Alizarin Red.

The toxicity of candidate dye-based photon reducing agents on mammaliancells was tested with wild-type Jurkat cells. Cells were incubated inmicrotiter plate assay wells at room temperature for 3 hours in thepresence of different concentrations of candidate dye-based photonreducing agents. Propidium iodide was then added to all wells of theassay plate, and the percentage of dead cells in each well wasestimated. Dead cells did not exclude propidium iodide.

FIG. 12 summarizes the results of candidate dye-based photon reducingagent toxicity testing. Over the concentrations tested only onecandidate dye-based photon reducing agent showed significantcytotoxicity over a three hour time period. Presumably at shorter timeperiods, the candidate dye-based photon reducing agents will have evenless of a cytotoxic effect.

Example 9 Testing of Dye-Based Photon Reducing Agents for Affects onGene Activation and Intracellular Enzyme Activity in Cell Based Assays

To further investigate the ability of candidate dye-based photonreducing agents to reduce undesired fluorescence in a cell-based assay,gene activation and intracellular enzyme activity in the presence of acandidate photon reducing agent was monitored as a function of thephoton reducing agent concentration. The candidate photon reducingagents were selected from a number of dyes based on their absorbancespectra and use with living systems. The following experimentsdemonstrate that dye-based photon reducing agents can be easily testedand selected for their compatibility with cell-based assays havingtranscriptional activity.

Jurkat cells were treated as described herein for CCF2 experiments. Thecells have a G-protein coupled receptor that can activate a responseelement controlling the transcription of beta-lactamase. Cells werestimulated with an agonist for the G-protein coupled receptor in thepresence of different concentrations of individual candidate dye-basedphoton reducing agents that were preincubated with the cells. Cells werethen incubated with CCF2/AM. Cells were subsequently evaluated forCCF2/AM loading, and conversion to CCF2, and for reporter geneexpression using a microtiter plate fluorimeter. Immediately beforefluorescence readout, the photon reducing agent, Schilling Red Food Dye,was added to all wells of the assay plate in order to normalize solutionfluorescence. Thus, these experiments were designed to investigate cellfunction in the presence candidate photon reducing agents.

FIG. 13 illustrates the ability of cells treated with candidatedye-based photon reducing agents to express beta-lactamase uponstimulation with agonist, load substrate and convert substrate. Directobservation of the cells also showed that the cells loaded substrate toits trapped form, as well as having beta-lactamase activity. 2-folddilutions of ESS dyes ranging from final concentrations of 0.039 mM(left) to 10 mM (right), with the exception of Patent Blue, which rangedfrom 0.022 mM (left) to 2.75 mM (right). Data is presented as a ratio ofratios ([agonist stimulated cells at emission wavelengths 460/530nm]/[unstimulated cells emission 460/530 nm]).

These results demonstrate that cells treated with candidate dye-basedphoton reducing agents can load and convert substrate to its trappedform and support G-protein coupled receptor activation and reporter geneexpression. Substrate loading and trapping indicates that the cellmembrane is intact during candidate photon reducing agent treatment.Substrate trapping also indicates that intracellular esterases aresufficiently active to convert CCF2/AM into its trapped form CCF2.G-protein coupled receptor activation, gene activation and genetranscription processes also remain active in the presence of candidatephoton reducing agents, as evidenced by beta-lactamase expression.Finally, beta-lactamase activity is sufficiently high in cells treatedwith candidate photon reducing agents to permit signal detectioncomparable to signal detection in the absence of candidate photonreducing agents. In some instances, signal over background frombeta-lactamase expressing cells were actually increased by the presenceof candidate photon reducing agents, suggesting that combinations ofphoton reducing reagents can actually provide superior results.

These experiments are quite rigorous in the testing of candidate photonreducing agents because the length of incubation with such photonreducing agents was approximately three hours and the photon reducingagents were added prior to gene activation and expression. In manyscreening and assay protocols, photon reducing agents can be added justprior to fluorescence detection, thereby minimizing the effects suchphoton reducing agents may have on the cells or assay.

Example 10 Dye-Based Photon Reducing Agent Sets Reduce UndesiredFluorescence in Cell Based Assays Better than a Single Dye-Based PhotonReducing Agent

To investigate the ability of dye-based photon reducing agent sets toreduce undesired fluorescence in a cell-based assay, dye-based photonreducing agent sets were compared with a single photon reducing agent inthe cell-based assays described herein. Dye-based photon reducing agentsets refer to at least two dye-based photon reducing agents. Thefollowing experiments demonstrate that dye-based photon reducing agentsets can be yield better cell based assay results, such as improvedsignal to noise ratios and are more robust at protecting againstundesired fluorescence.

The photon reducing agent considered for use in the sets were selectedfrom a number of dyes based on the following criteria: solubility inaqueous solution, having sufficiently high molar extinction coefficient,having low toxicity to mammalian cells, and not interfering with geneexpression, substrate loading, and substrate conversion. The followingdyes were selected: Tartrazine, Naphthol Yellow, Chromotrope F8,Chromazurol S, Patent Blue, Chromotrope 2R, Acid Fuchsin, and Acid Red37.

From this list of dyes, two mixtures were created, based on theabsorbance spectra of the dyes. Dyes selection was based on which dyesets would absorb solution fluorescence over the range of wavelengthsfor CCF2 excitation, coumarin emission and fluorescein emission. The twomixtures were called “ESS Mix 1” and “ESS Mix 2.” ESS Mix 1 was: 100 mMTartrazine, 100 mM Chromotrope 2R, and 100 mM Acid Fuchsin. ESS Mix 2was: 40 mM Tartrazine, 60 mM Acid Red 37, and 40 mM Acid Fuchsin.

The ESS dye mixtures were assayed for appropriate concentrations foroptimal use in the homogeneous assay for beta-lactamase, as describedherein. First, dilutions of ESS Mix 1 and ESS Mix 2 were tested usingRed Food Dye as a control. In all cases tested, ESS Mix 1 and ESS Mix 2improved the fluorescence readout more than Red Food Dye. Subsequentexperiments were used to evaluate lower concentrations of the ESSmixtures in order to effectively titrate the amount of the ESS mixturesneeded for optimal assay performance.

After the initial set of ESS mixture testing, one more variation of dyeswas made. The third ESS mixture was given the name “Tararaf” (an acronymfor Tartrazine, Acid Red 37 and Acid Fuchsin). Tararaf is: 50 mMTartrazine, 60 mM Acid Red 37, 40 mM Acid Fuchsin.

Tararaf was compared to ESS Mix 1 and ESS Mix 2, as well as to theindividual components of Tararaf and Red Food Dye, in cell-based assaysusing CCF2. Tararaf improved the fluorescence readout more than each ofthe individual components of the mixture did.

FIG. 14 shows the results of these experiments. These resultsdemonstrate that photon reducing agent sets can improve signals fromcell-based assays compared to either single photon reducing agents or nophoton reducing agents.

Publications

All publications, including patent documents, world wide web sites andscientific articles, referred to in this application are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication were individually incorporated by reference.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A method of reducing undesired light emission from a sample, comprising: contacting a sample in need of reducing undesired light with at least one photon reducing agent, wherein said at least one photon reducing agent is substantially membrane impermeant, wherein said sample comprises a membrane compartment, wherein said membrane compartment includes at least one photon producing agent and said at least one photon reducing agent is in an aqueous solution that contacts an outer surface of said membrane compartment, wherein said at least one photon reducing agent does not specifically bind to said membrane compartment, and detecting an optical signal from said at least one photon producing agent.
 2. The method of claim 1, wherein said at least one photon reducing agent has an absorption spectra that overlaps with the absorption, emission or excitation spectrum of said at least one photon producing agent.
 3. The method of claim 2, wherein said at least one photon producing agent exhibits fluorescence resonance energy transfer with said at least one photon reducing agent.
 4. The method of claim 1, wherein said sample comprises at least two photon reducing agents.
 5. The method of claim 1, wherein said membrane compartment comprises at least one living cell.
 6. The method of claim 1, wherein said at least one photon reducing agent is selected from the group consisting of a collisional quencher, a particulate, an absorption quencher, a FRET quencher and a dark complex.
 7. The method of claim 1, wherein said at least one photon reducing agent is a dye.
 8. The method of claim 1, wherein said detecting comprises detecting a fluorescence signal.
 9. The method of claim 8, wherein said at least one photon producing agent is produced from a precursor molecule that is a substrate for an esterase.
 10. The method of claim 9, wherein said at least one photon producing agent detects the presence of an ion inside said membrane compartment.
 11. The method of claim 9, wherein said at least one photon producing agent is a fluorescent protein.
 12. The method of claim 9, wherein said at least one photon producing agent detects voltage across a membrane of said membrane compartment.
 13. The method of claim 8, wherein the detecting is by fluorescence activated cell sorting.
 14. The method of claim 1, wherein said membrane compartment is within a droplet.
 15. A composition of matter, comprising: a) a membrane compartment, wherein said membrane compartment comprises at least one photon producing agent, and b) an aqueous solution with at least one photon reducing agent, wherein said aqueous solution is in contact with an outer surface of said membrane compartment, wherein said at least one photon reducing agent is substantially membrane impermeant and does not specifically bind to said membrane compartment, wherein said at least one photon reducing agent has an absorption spectrum that overlaps with the absorption, emission or excitation spectrum of said at least one photon producing agent, and wherein said at least one photon reducing agent is present in said aqueous solution at a concentration sufficient to reduce light emitted from said aqueous solution by at least 10% compared to the light emitted of said aqueous solution in the absence of said at least one photon reducing agent.
 16. The composition of matter of claim 15, wherein said at least one photon reducing agent has an absorption spectrum that overlaps with the emission or excitation spectrum of said at least one photon producing agent.
 17. The composition of matter of claim 15, wherein said at least one photon producing agent can transfer energy to said at least one photon reducing agent.
 18. The composition of matter of claim 15, wherein said membrane compartment is within a droplet.
 19. The composition of matter of claim 15, wherein said membrane compartment comprises at least one living cell. 